Image flow cytometer based on optical fiber micro-fluidic chip

Abstract

The invention provides an image flow cytometer based on an optical fiber micro-fluidic chip, which comprises an optical fiber micro-fluidic chip, the optical fiber micro-fluidic chip comprises a plurality of excitation areas at different positions and corresponding optical fiber sockets, the optical fiber sockets are used for inserting corresponding optical fibers, the optical fibers are connected with corresponding optical fiber lasers, and the optical fiber lasers are connected with the optical fiber micro-fluidic chip. The fiber laser is used for transmitting corresponding excitation light to the excitation area through optical fiber conduction; when the cell samples sequentially flow through the excitation areas, the cell samples are sequentially excited by the corresponding excitation light to generate independent fluorescence signals of different spectrum bands, and when the bright field light source irradiates the imaging detection areas where the excitation areas are located, bright field signals are generated; independent fluorescence signals and bright field signals of different spectrum bands are synchronously collected through a fluorescence bright field synchronous collection light path, and independent multi-spectrum-band fluorescence images and bright field images are obtained. According to the invention, independent multispectral fluorescence images and bright field images can be obtained at the same time without using a high-cost laser and matched laser calibration, focusing optical elements and optical light splitting elements, and the complexity and the cost are relatively low.

Description

Technical Field

[0001] The invention relates to the technical field of image flow cytometers, and in particular to an image flow cytometer based on an optical fiber microfluidic chip. Background Art

[0002] Image flow cytometer is an instrument that combines flow cytometry and microscopic imaging technology for high-throughput, quantitative and qualitative analysis of cells and particles. Compared with traditional flow cytometers, image flow cytometers can not only detect fluorescent signals, but also capture images of each cell, providing more information for cell morphology analysis. Similar to traditional flow cytometers, it is also composed of three major parts: fluid system, optical system and electronic system.

[0003] The existing image flow cytometer optical system uses multiple lasers to emit lasers of different wavelengths, which need to be precisely calibrated by optical components and focused on the detection area to excite cell fluorescence. Multiple lasers of different wavelengths are expensive, and the calibration and focusing of multiple lasers of different wavelengths require multiple optical components, making the optical system and adjustment relatively complex. When cells flow through the detection window, they are stimulated to emit corresponding multi-spectral fluorescence signals. Since the multi-spectral fluorescence signals are coupled and overlapped in space, they need to be split into spatially independent signals using optical spectrometers before they can be recorded in different areas of the camera imaging surface to distinguish between different spectral fluorescence channels. The introduction of spectrometers further increases the complexity and cost of the optical system. In addition, in order to obtain fluorescence images and bright field images at the same time, the existing image flow cytometer needs to focus the excitation light and bright field light of multiple wavelengths on the same position of the cell flow channel at the same time. A large number of optical components are used, and the adjustment precision requirements are high. The existing image flow cytometer uses sheath fluid flow to make cells flow in a single column. Therefore, in addition to the cell sample liquid, a buffer solution needs to be added as the sheath fluid. The sample liquid and the buffer solution also require a specific pressure to form the sheath fluid flow, which is inconvenient to use. At the same time, the flow channel of the sheath fluid needs to be cleaned with a cleaning fluid after each use, which increases the complexity of the fluid system.

[0004] That is, existing image flow cytometers cannot simultaneously obtain independent multi-spectral fluorescence images and bright field images in a low-cost and low-complexity manner without using high-cost lasers and supporting laser calibration, focusing optical elements, and optical splitting elements.

[0005] To address the above problems, no effective solution has been proposed yet. Summary of the invention

[0006] The embodiments of this specification provide an image flow cytometer based on a fiber optic microfluidic chip to solve the problem that the prior art cannot simultaneously obtain independent multi-spectral fluorescence images and bright field images in a low-cost and low-complexity manner.

[0007] In order to solve the above technical problems, the embodiment of this specification provides an image flow cytometer based on an optical fiber microfluidic chip, including an optical fiber microfluidic chip and a fluorescence bright field synchronous collection optical path, wherein the optical fiber microfluidic chip includes a plurality of excitation regions at different positions and optical fiber sockets corresponding to the excitation regions, wherein the excitation regions are located in an imaging detection region, wherein the optical fiber sockets are used to insert corresponding optical fibers, wherein the optical fibers are connected to an optical fiber laser of a corresponding wavelength, wherein the optical fiber laser is used to transmit corresponding excitation light to the excitation regions through optical fiber conduction;

[0008] When the cell sample flows through the excitation zones at different positions in sequence, it is excited by the corresponding excitation light in sequence, generating independent fluorescence signals of different spectral bands, and when the bright field light source irradiates the imaging detection area, a bright field signal is generated;

[0009] The independent fluorescence signals of different spectral bands and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path to obtain independent multi-spectral fluorescence images and bright field images.

[0010] In some embodiments, the fiber optic microfluidic chip further comprises a cell flow channel, in which the imaging detection area exists, and the cell sample flows from a cell sample inlet of the cell flow channel through the cell imaging detection area and flows out from a cell sample outlet of the cell flow channel.

[0011] In some embodiments, the fiber optic microfluidic chip enables the cell sample to form a single-file flow or a nearly single-file flow.

[0012] In some embodiments, the multiple excitation zones at different positions include a first excitation zone located on the left side of the imaging detection zone, a second excitation zone located in the middle of the imaging detection zone, and a third excitation zone located on the right side of the imaging detection zone. The optical fiber sockets corresponding to the excitation zones include a first optical fiber socket corresponding to the first excitation zone, a second optical fiber socket corresponding to the second excitation zone, and a third optical fiber socket corresponding to the third excitation zone. The first optical fiber socket is inserted into the first optical fiber, the second optical fiber socket is inserted into the second optical fiber, and the third optical fiber socket is inserted into the third optical fiber.

[0013] In some embodiments, the first optical fiber is connected to a fiber laser with a wavelength of 405 nm, the second optical fiber is connected to a fiber laser with a wavelength of 488 nm, and the third optical fiber is connected to a fiber laser with a wavelength of 647 nm.

[0014] In some embodiments, the bright field light source includes LED lights.

[0015] In some embodiments, the fluorescence bright field synchronous collection light path includes a fluorescence light path and a bright field light path, the fluorescence light path includes a 40X objective lens, a reflective transmission lens, a first condenser, a first filter, and a fluorescence camera in sequence, and the bright field light path includes a 40X objective lens, a reflective projection lens, a second condenser, a second filter, and a bright field camera in sequence.

[0016] In some embodiments, the independent fluorescence signals of different spectral bands and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path, including:

[0017] The independent fluorescence signals of different spectral bands are collected by a 40X objective lens, imaged by a fluorescence camera after passing through a reflection transmission mirror, a first condenser, and a first filter, to obtain an independent multi-spectral fluorescence image. At the same time, the bright field signal is collected by a 40X objective lens, imaged by a bright field camera after passing through a reflection transmission mirror, a second condenser, and a second filter, to obtain a bright field image.

[0018] In some embodiments, the chip thickness of the optical fiber microfluidic chip is controllable, and when the chip thickness is controlled within a preset range, the obtained bright field image is a high-definition bright field image.

[0019] In some embodiments, the fiber optic microfluidic chip can be replaced.

[0020] The embodiment of this specification provides an image flow cytometer based on a fiber optic microfluidic chip, including a fiber optic microfluidic chip and a fluorescence bright field synchronous collection optical path, wherein the fiber optic microfluidic chip includes multiple excitation areas at different positions and fiber optic sockets corresponding to the excitation areas, wherein the excitation areas are located in an imaging detection area, wherein the fiber optic socket is used to insert a corresponding optical fiber, wherein the optical fiber is connected to a fiber optic laser of a corresponding wavelength, wherein the fiber optic laser is used to transmit a corresponding excitation light to the excitation area through optical fiber conduction; when the cell sample flows through the excitation areas at multiple different positions in sequence, it is sequentially excited by the corresponding excitation light, generating independent fluorescence signals of different spectral bands, and generating a bright field signal when a bright field light source is irradiated to the imaging detection area; the independent fluorescence signals of different spectral bands and the bright field signal are synchronously collected through the fluorescence bright field synchronous collection optical path, and an independent multi-spectral fluorescence image and a bright field image are obtained. In the embodiment of this specification, by using a low-cost fiber optic laser as an excitation light source, the cost of the light source is greatly reduced. By setting up a fiber optic microfluidic chip including multiple excitation zones at different positions, the excitation zones at different positions have corresponding fiber optic sockets, the fiber optic sockets can be inserted with corresponding optical fibers, and the optical fibers are connected to fiber optic lasers of corresponding wavelengths, so that the excitation light emitted by the fiber optic lasers of corresponding wavelengths can be transmitted through the optical fiber and irradiated on the excitation zones at different positions, without the need to use precision optical elements to calibrate and focus the lasers emitted by multiple lasers of different wavelengths as in traditional image flow cytometers, thereby reducing the complexity of the optical system. In addition, the excitation light irradiates the excitation zones at different positions, so that the cell samples flowing in the fiber optic microfluidic chip can be excited at different times and different positions, generating independent fluorescence signals of different spectrum bands, which are independent in space and time, and do not need to add optical spectroscopic elements to decouple the fluorescence signals of different spectrum bands, further reducing the cost and complexity of the optical system. Furthermore, when the bright field light source irradiates the imaging detection area, a bright field signal is generated, and the independent fluorescence signals and bright field signals of different spectral bands are synchronously collected through the fluorescence bright field synchronous collection optical path, so that independent multi-spectral fluorescence images and bright field images can be obtained at the same time, without the need to use a large number of optical components. The complexity and adjustment difficulty of the optical system are much lower than those of existing image flow cytometers. Through the above scheme, independent multi-spectral fluorescence images and bright field images can be obtained at the same time without the need to use high-cost lasers and supporting laser calibration, focusing optical components, and optical spectrometers, with low complexity and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0022] Figure 1 is a schematic diagram of the structure of the optical fiber microfluidic chip provided in the embodiments of this specification;

[0023] Figure 2 It is a schematic diagram of the structure of an image flow cytometer based on a fiber optic microfluidic chip provided in an embodiment of this specification. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this specification, not all of the embodiments. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this specification.

[0025] Image flow cytometer combines traditional flow cytometer and microscopic imaging technology, which can provide statistical data of cell groups and obtain images of single cells, thus providing information on cell morphology, cell structure and subcellular signal distribution. Similar to traditional flow cytometer, it is also composed of three major parts: liquid flow system, optical system and electronic system.

[0026] Existing image flow cytometers have the following problems:

[0027] 1. Optical system

[0028] Existing image flow cytometers use high-cost lasers of multiple different wavelengths to stimulate the fluorescence signals of cells, and require the use of precision optical components to calibrate and focus the lasers of multiple wavelengths on the cells, which requires high accuracy and complex adjustments. At the same time, the multi-spectral fluorescence signals stimulated by the cells overlap in space, and the overlapping multi-spectral fluorescence signals need to be split with the help of a spectroscopic element to achieve the shooting of fluorescence channels of different wavelengths, further increasing the complexity of the optical system and the difficulty of adjustment.

[0029] 2. Liquid flow system

[0030] Existing image flow cytometer fluid systems use sheath fluid flow to form a single-file flow of cells. Therefore, in addition to the cell sample solution, a buffer solution needs to be added as the sheath fluid. The sample solution and the buffer solution require a specific pressure to form the sheath fluid flow, which is complicated to use. At the same time, the sheath fluid flow channel needs to be cleaned with a cleaning solution after each use, which increases the maintenance steps.

[0031] In view of the above problems existing in the existing methods and the specific reasons for the above problems, this application considers introducing an image flow cytometer based on a fiber optic microfluidic chip. It does not need to use high-cost lasers and supporting laser calibration. Focusing optical elements can stimulate independent fluorescence signals of different spectral bands of cell samples, and the fluorescence signals are independent in space and time. Independent multi-spectral fluorescence images can be obtained without introducing additional spectroscopic elements, which greatly reduces the complexity and cost of the optical system. The laser part is directly transmitted to the imaging detection area using optical fiber, and no additional optical adjustment is required. It only needs to focus the bright field light source on the imaging detection area to achieve simultaneous imaging of fluorescent images and bright field images. The optical system has low complexity and simple adjustment, which can effectively solve the problems of high complexity and difficulty in adjustment of the existing image flow cytometer optical system. In addition, there is no need to use sheath liquid flow. Based on the fiber optic microfluidic chip, cells can be formed into a single-line flow or a nearly single-line flow, which can avoid the use of sheath liquid buffer and does not require cleaning, reducing the complexity and cost of the liquid flow system.

[0032] See also Figure 1 As shown, the image flow cytometer of the above-mentioned fiber optic microfluidic chip may include a fiber optic microfluidic chip and a fluorescence bright field synchronous collection optical path, the fiber optic microfluidic chip includes a plurality of excitation regions at different positions and fiber optic sockets corresponding to the excitation regions, the excitation regions are located in the imaging detection area, the fiber optic sockets are used to insert corresponding optical fibers, the optical fibers are connected to fiber optic lasers of corresponding wavelengths, and the fiber optic lasers are used to emit corresponding excitation light to the excitation regions through optical fiber conduction;

[0033] When the cell sample flows through the excitation zones at different positions in sequence, it is excited by the corresponding excitation light in sequence, generating independent fluorescence signals of different spectral bands, and when the bright field light source irradiates the imaging detection area, a bright field signal is generated;

[0034] The independent fluorescence signals of different spectral bands and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path to obtain independent multi-spectral fluorescence images and bright field images.

[0035] In this embodiment, the cell sample can be dyed with a fluorescent dye and made into a uniform cell suspension, and the cell sample can flow in a single column or approximately in a single column in the cell flow channel of the optical fiber microfluidic chip. The optical fiber laser of the corresponding wavelength can emit an excitation light of the corresponding wavelength to excite the fluorescent dye in the cell sample and generate a fluorescent signal. The excitation areas at the above-mentioned multiple different positions can have corresponding optical fiber sockets, and the optical fiber sockets can be inserted into the corresponding optical fibers, and the optical fibers can be connected to the optical fiber lasers of the corresponding wavelengths, so that the corresponding excitation light emitted by the optical fiber laser can be transmitted through the optical fiber to irradiate the excitation areas at different positions, so that the cell samples flowing in the optical fiber microfluidic chip are excited at different positions and at different times, and independent fluorescence signals of different spectral bands are generated in time and space. There is no need to use precision optical elements to calibrate and focus the lasers emitted by multiple lasers of different wavelengths like traditional image flow cytometers, which reduces the complexity of the optical system, and there is no need to add optical spectroscopic elements to decouple the fluorescence signals of different spectral bands, further reducing the cost and complexity of the optical system. At the same time, when the bright field light source illuminates the imaging detection area, a bright field signal is generated, and independent fluorescence signals and bright field signals of different spectral bands are synchronously collected through the fluorescence bright field synchronous collection optical path, so that independent multi-spectral fluorescence images and bright field images can be obtained at the same time. There is no need to use a large number of optical components. The complexity and adjustment difficulty of the optical system are much lower than those of existing image flow cytometers.

[0036] Through the above embodiments, independent multi-spectral fluorescence images and bright field images can be obtained simultaneously without using high-cost lasers and supporting laser calibration, focusing optical elements, and optical splitting elements, with low complexity and cost.

[0037] In some embodiments, the above-mentioned fiber optic microfluidic chip may also include a cell flow channel, which may include a cell sample inlet and a cell sample outlet. The imaging detection area exists in the cell flow channel, and the cell sample flows from the cell sample inlet of the cell flow channel through the cell imaging detection area and flows out from the cell sample outlet of the cell flow channel.

[0038] In some embodiments, the fiber optic microfluidic chip enables the cell sample to form a single-file flow or a nearly single-file flow.

[0039] Specifically, the flow direction of the cell sample (referred to as the cell flow direction) is from the cell sample inlet to the cell sample outlet, passing through the imaging detection area. Since the optical fiber microfluidic chip uses the deterministic lateral displacement principle to make the cells flow in a single column or approximately in a single column, the use of sheath liquid buffer can be avoided, solving the problem that the existing image flow cytometer needs to use sheath liquid flow to make the cells flow in a single column, which causes the complexity and high cost of the liquid flow system. Among them, the cell sample inlet can be on the left side of the optical fiber microfluidic chip, and the cell sample outlet can be on the right side of the optical fiber microfluidic chip.

[0040] In some embodiments, the above-mentioned multiple excitation zones at different positions may include a first excitation zone located on the left side of the imaging detection zone, a second excitation zone located in the middle of the imaging detection zone, and a third excitation zone located on the right side of the imaging detection zone. The optical fiber sockets corresponding to the excitation zones include a first optical fiber socket corresponding to the first excitation zone, a second optical fiber socket corresponding to the second excitation zone, and a third optical fiber socket corresponding to the third excitation zone. The first optical fiber socket is inserted into the first optical fiber, the second optical fiber socket is inserted into the second optical fiber, and the third optical fiber socket is inserted into the third optical fiber.

[0041] In some embodiments, the first optical fiber is connected to a fiber laser with a wavelength of 405 nm, the second optical fiber is connected to a fiber laser with a wavelength of 488 nm, and the third optical fiber is connected to a fiber laser with a wavelength of 647 nm.

[0042] For details, see Figure 1 As shown, the first excitation area (405nm excitation area) can be A, the second excitation area (488nm excitation area) can be B, and the third excitation area (647nm excitation area) can be C. A can be located on the left side of the imaging detection area of ​​the optical fiber microfluidic chip, B can be located in the middle of the imaging detection area of ​​the optical fiber microfluidic chip, and C can be located on the right side of the imaging detection area of ​​the optical fiber microfluidic chip. The first optical fiber socket corresponding to A can be inserted with the first optical fiber, and the first optical fiber can be connected to a 405nm wavelength optical fiber laser, so that the 405nm wavelength excitation light emitted by the 405nm wavelength optical fiber laser is transmitted through the first optical fiber and irradiated on A. The second optical fiber socket corresponding to B can be inserted with the second optical fiber, and the second optical fiber can be connected to a 488nm wavelength optical fiber laser, so that the 488nm wavelength excitation light emitted by the 488nm wavelength optical fiber laser is transmitted through the second optical fiber and irradiated on B. The third optical fiber socket corresponding to C can be inserted with a third optical fiber, and the third optical fiber can be connected to a 647nm wavelength optical fiber laser, so that the 647nm wavelength excitation light emitted by the 647nm wavelength optical fiber laser is transmitted and irradiated on C through the third optical fiber.

[0043] After the cell sample enters the internal flow channel of the fiber optic microfluidic chip from the cell sample entrance, the cell sample flows to the right and gradually flows to the imaging detection area. When the cell sample flows through the imaging detection area, it will pass through the excitation area A in sequence and be excited by the excitation light of 405nm wavelength, pass through the excitation area B and be excited by the excitation light of 488nm wavelength, and pass through the excitation area C and be excited by the excitation light of 647nm wavelength. Since the cell sample is excited in three different positions A, B, and C in sequence, the different spectral fluorescence signals excited by the three excitation lights do not overlap, but are separated in space and time. Therefore, there is no need to use optical elements to split the fluorescence signals of different spectral bands like traditional image flow cytometers. The multi-spectral fluorescence signals excited in sequence at different positions can be directly recorded on the fluorescence camera to obtain independent images of each fluorescence channel.

[0044] It should be noted that the number of the above-mentioned excitation zones and corresponding optical fiber sockets can be changed according to actual needs, and spare sockets can be set according to actual needs. The above-mentioned wavelengths can be changed according to actual needs, and this manual does not make specific limitations on this.

[0045] In some embodiments, the bright field light source includes LED light.

[0046] In some embodiments, the above-mentioned fluorescence bright field synchronous collection light path includes a fluorescence light path and a bright field light path. The fluorescence light path includes a 40X objective lens, a reflective transmission lens, a first condenser, a first filter, and a fluorescence camera in sequence. The above-mentioned bright field light path includes a 40X objective lens, a reflective projection lens, a second condenser, a second filter, and a bright field camera in sequence.

[0047] In some embodiments, the independent fluorescence signals of different spectral bands and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path. When specifically implemented, the following steps may be included:

[0048] The independent fluorescence signals of different spectral bands are collected by a 40X objective lens, imaged by a fluorescence camera after passing through a reflection transmission mirror, a first condenser, and a first filter, to obtain an independent multi-spectral fluorescence image. At the same time, the bright field signal is collected by a 40X objective lens, imaged by a bright field camera after passing through a reflection transmission mirror, a second condenser, and a second filter, to obtain a bright field image.

[0049] For details, see Figure 2As shown, when the cell sample flows through the A, B, and C excitation areas in the imaging detection area, it is excited by the excitation light irradiated in the A, B, and C excitation areas in turn, and the independent fluorescence signals of different spectral bands are collected by the 40X objective lens, and imaged on the fluorescence camera after passing through the reflection transmission mirror, the first condenser, and the second filter, thereby obtaining three fluorescence channel images of the cell sample, that is, obtaining an independent multi-spectral fluorescence image. A bright field light source, such as an LED light, can be directly irradiated on the imaging detection area, collected by the 40X objective lens after passing through the cell sample, and imaged on the bright field camera after being reflected by the reflection transmission mirror and passing through the second focusing mirror and the second filter, thereby obtaining a bright field image of the cell sample. Among them, the LED light and the bright field camera are important components of bright field (microscope) imaging. When the bright field camera and the fluorescence camera work at the same time, the bright field image of the cell and the independent multi-spectral fluorescence image (or multi-channel fluorescence image) can be synchronously captured.

[0050] By adjusting the bright field light source to illuminate the imaging detection area, multi-channel fluorescence images and bright field images of the cell sample can be obtained simultaneously without using a large number of optical elements. The complexity and cost of obtaining multi-channel fluorescence images and bright field images at the same time are low.

[0051] In some embodiments, the chip thickness of the optical fiber microfluidic chip is controllable, and when the chip thickness is controlled within a preset range, the obtained bright field image is a high-definition bright field image.

[0052] In some embodiments, the optical fiber microfluidic chip can be replaced.

[0053] Since the thickness of the fiber optic microfluidic chip is controllable, when the chip thickness is controlled within a reasonable range (i.e., the preset range mentioned above), the light transmission effect of its cell flow channel is better than that of the cell flow channel used in the existing image flow cytometer, thereby obtaining a high-definition bright field image that is far superior to the existing image flow cytometer. Since the fiber optic microfluidic chip used is replaceable and does not require liquid path cleaning, the complexity and cost of the liquid flow system can be further reduced.

[0054] In some embodiments, the bright field camera may use a CCD or CMOS camera to capture the image of each cell. The fluorescence camera may use a high-sensitivity fluorescence camera to capture the fluorescence image of the cell, which usually includes multiple fluorescence channels.

[0055] In some embodiments, after obtaining independent multi-spectral fluorescence images and bright field images, fluorescence intensity analysis, image processing, cell classification, etc. can be performed.

[0056] In some embodiments, the above-mentioned fiber optic microfluidic chip can transmit the excitation light of the fiber optic laser to the cell sample through the fiber optic transmission by inserting the optical fiber, without the need to use optical components to calibrate and focus on the cell excitation area, and the system construction and adjustment are simple and convenient. In addition, the use of low-cost fiber optic lasers as the excitation light source greatly reduces the cost of the light source.

[0057] Based on the above embodiments, this specification uses low-cost fiber lasers as excitation light sources, which greatly reduces the cost of laser light sources; lasers of multiple wavelengths are transmitted to irradiate cell samples by inserting optical fibers, and lasers of multiple wavelengths are transmitted through optical fibers to irradiate the imaging detection area in the optical fiber microfluidic chip to excite cell samples in the excitation area at different positions in the imaging detection area, without the need to use optical elements to calibrate and focus lasers of multiple wavelengths on cells in the imaging detection area like traditional image flow cytometers; excitation lights of different wavelengths excite cell fluorescence signals at different positions, and different spectral bands are obtained Fluorescence signals are independent in space and time, and there is no need to add optical spectrometers in the fluorescence light path to decouple fluorescence signals in different spectral bands. With the help of fiber-optic microfluidic chips, fluorescence imaging and bright-field imaging can be jointly imaged, and image synchronization can be achieved, which greatly simplifies the complexity and cost of the optical system. The thickness of the fiber-optic microfluidic chip is controllable. If it is made thinner, excellent light transmittance can be obtained, and the clarity of the bright-field image obtained is better than that of commercial products. The cell flow channel inside the fiber-optic microfluidic chip integrates a chip structure based on the principle of deterministic lateral displacement, which allows cells to flow in a single row / column, without the need for sheath flow, reducing the complexity of the flow system.

[0058] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other. Each embodiment focuses on the differences from other embodiments.

[0059] Although the present specification is described through embodiments, those skilled in the art will appreciate that there are many modifications and changes to the present specification without departing from the spirit of the present specification, and it is intended that the appended claims include these modifications and changes without departing from the spirit of the present specification.

Claims

1. An image flow cytometer based on an optical fiber microfluidic chip, characterized in that: It includes an optical fiber microfluidic chip and a fluorescence bright field synchronous collection optical path, wherein the optical fiber microfluidic chip includes a plurality of excitation regions at different positions and optical fiber sockets corresponding to the excitation regions, wherein the excitation regions are located within an imaging detection region, wherein the optical fiber sockets are used to insert corresponding optical fibers, wherein the optical fibers are connected to an optical fiber laser of a corresponding wavelength, and wherein the optical fiber laser is used to transmit corresponding excitation light to the excitation regions through optical fiber conduction; When the cell sample flows through the excitation zones at different positions in sequence, it is excited by the corresponding excitation light in sequence, generating independent fluorescence signals of different spectral bands, and when the bright field light source irradiates the imaging detection area, a bright field signal is generated; The independent fluorescence signals of different spectral bands and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path to obtain independent multi-spectral fluorescence images and bright field images.

2. The image flow cytometer according to claim 1, characterized in that: The optical fiber microfluidic chip also includes a cell flow channel, in which the imaging detection area exists, and the cell sample flows from the cell sample inlet of the cell flow channel through the cell imaging detection area and flows out from the cell sample outlet of the cell flow channel.

3. The image flow cytometer according to claim 1, characterized in that: The optical fiber microfluidic chip enables the cell sample to form a single-file flow or a nearly single-file flow.

4. The image flow cytometer according to claim 1, characterized in that: The multiple excitation zones at different positions include a first excitation zone located on the left side of the imaging detection zone, a second excitation zone located in the middle of the imaging detection zone, and a third excitation zone located on the right side of the imaging detection zone. The optical fiber sockets corresponding to the excitation zones include a first optical fiber socket corresponding to the first excitation zone, a second optical fiber socket corresponding to the second excitation zone, and a third optical fiber socket corresponding to the third excitation zone. The first optical fiber socket is inserted into the first optical fiber, the second optical fiber socket is inserted into the second optical fiber, and the third optical fiber socket is inserted into the third optical fiber.

5. The image flow cytometer according to claim 4, characterized in that: The first optical fiber is connected to a fiber laser with a wavelength of 405 nm, the second optical fiber is connected to a fiber laser with a wavelength of 488 nm, and the third optical fiber is connected to a fiber laser with a wavelength of 647 nm.

6. The image flow cytometer according to claim 1, characterized in that: The bright field light source includes LED light.

7. The image flow cytometer according to claim 1, characterized in that: The fluorescence bright field synchronous collection light path includes a fluorescence light path and a bright field light path. The fluorescence light path includes a 40X objective lens, a reflection transmission mirror, a first condenser, a first filter, and a fluorescence camera in sequence. The bright field light path includes a 40X objective lens, a reflection projection mirror, a second condenser, a second filter, and a bright field camera in sequence.

8. The image flow cytometer according to claim 7, characterized in that: The independent fluorescence signals of different spectral segments and the bright field signals are synchronously collected through the fluorescence bright field synchronous collection optical path, including: The independent fluorescence signals of different spectral bands are collected by a 40X objective lens, imaged by a fluorescence camera after passing through a reflection transmission mirror, a first condenser, and a first filter, to obtain an independent multi-spectral fluorescence image. At the same time, the bright field signal is collected by a 40X objective lens, imaged by a bright field camera after passing through a reflection transmission mirror, a second condenser, and a second filter, to obtain a bright field image.

9. The image flow cytometer according to claim 1, characterized in that: The chip thickness of the optical fiber microfluidic chip is controllable. When the chip thickness is controlled within a preset range, the obtained bright field image is a high-definition bright field image.

10. The image flow cytometer according to claim 1, characterized in that: The optical fiber microfluidic chip can be replaced.

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